Expanding luciferase reporter systems for cell-free protein expression

Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.


Figure S1
Figure S1 Luminescence measurements with emission filters. Luminescence signals were measured with emission filters at the wavelength appropriate for each luciferase. The substrate mixtures were prepared as "All" (D-luciferin, Coelenterazine h, hispidin, octanaldehyde, Mg + , ATP, NADPH, FMN) or "All minus one" that contains all except one that a substrate is supposed to react with a tested luciferase. The assay was performed by mixing substrates with TXTL expressing each luciferase. The emission filter at 300 nM was used for the negative control. The graphs show means with error bars that signify SEM (n = 3).

Figure S2
Background signals of the substrate specificity assay. The luciferases used in the reaction are indicated as FLuc, RLuc, H3H-Luz, or LuxAB-Fre. Control stands for reaction without enzyme expression. Substrates in the reaction were indicated as "All" (D-luciferin, coelenterazine h, hispidin, octanaldehyde) or "All minus one", that one is the substrate supposed to react with the tested luciferase. The graphs show means with error bars that signify SEM (n = 3).

Figure S3
Western blot gels from the substrate specificity reactions. The loaded samples directly came from the reactions shown in Fig. 2B, indicating that the luciferase enzymes were expressed in the reactions. 15 μl of TxTL was loaded on each lane. FLuc and RLuc samples were fractionated on a 7.5% gel for 70 minutes at 100V. NanoLuc and H3H-Luz samples were fractionated on a 12% gel for 80 minutes at 100V. M, BLUEstain 2 Protein ladder, 5-245 kDa (Goldbio, P008-500); substrate names, a substrate that is contained in the reaction; T7RNAP, N-terminal His-tagged T7 RNA polymerase; FLuc, firefly luciferase with C-terminal His-tag; RLuc, Renilla luciferase with C-terminal His-tag; NanoLuc, NanoLuc luciferase with C-terminal His-Tag; H3H, hispidin-3-hydroxylase with C-terminal His-tag; Luz, fungi luciferase with C-terminal His-tag.   After expressing LuxABCDE+Fre or LuxAB+Fre in TXTL, 1 mM octanaldehyde was added as the substrate (time = 0). The luminescence was measured after 0.5, 1, 6, 8 hours. LuxABCDE-Fre, a reaction with TXTL expressing LuxAB-Fre and LuxCDE; LuxAB-Fre, a reaction with TXTL expressing LuxAB-Fre; Control, reaction with TXTL without enzyme expression. The graphs show means with error bars that signify SEM (n = 3). In this experiment, the luminescence was the highest at 0.5 hours, while the starting time (t = 0) was the highest in the similar experiment using decanoic acid (Fig. 3C). This luminescence delay was caused probably because we held mixed reactions on ice for 10 minutes, during the recovery of the connection error between a plate reader and a computer. We think this holding on ice cooled the reactions and caused the delay reaching the maximum luminescence.    Western blot of Luz-GFP fusion proteins. 15 μl of TXTL was loaded on each lane and fractionated on a 7.5% gel for 60 minutes at 100V. The lane labeled with M stands for BLUEstain 2 Protein ladder, 5-245 kDa (Goldbio, P008-500). The protein names used for lane labels represent the fusion protein constructs from N-terminal to C-terminal order. His, His-Tag; GFP, enhanced green fluorescent protein; 2GS, two GS-linker sequence (GGGGS) repeats; 3GS, three GS-linker sequence repeats; T7 RNAP, T7 RNA polymerase. The bottom image is the original data.

Figure S10
Figure S10 Fluorescence generated from eGFP-luciferase fusion proteins (Luz) with extended GSlinker. All fusion proteins were expressed in TXTL at 30 ˚C for 8 hours, followed by fluorescence measurement. 19 μl of TXTL was used for the measurement. eGFP and luciferases are linked through 3x GS-linker (GGGGS.) Control stands for a reaction without protein expression.

Figure S11
Figure S11 LuxA and LuxB capability as fusion proteins.
(A and B) eGFP fluorescence measurement in TXTL. Fusion LuxA and LuxB were expressed in TXTL at 30°C for 8 hours, followed by the fluorescence measurement. (C) Luminescence measurement with all the combinations of LuxA and LuxB fusion constructs. LuxA and LuxB were expressed in TXTL, and then 1 mM Octanaldehyde was added, followed by luminescence measurement. Green shading behind the construct images indicates that the constructs fluorescence when expressed in TXTL. N-GFP, N-terminal eGFP fusion luciferase; C-GFP, C-terminal eGFP fusion luciferase; Control, reaction without enzyme expression.  All fusion proteins were expressed in TXTL at 30 ˚C for 8 hours, followed by fluorescence measurement. 19 μl of TXTL was used for the measurement. eGFP and luciferases are linked through 3x GS-linker (GGGGS.) Control stands for a reaction without protein expression. 15 μl of TXTL was loaded on each lane and fractionated on 7.5% gel for 60 minutes at 100V. The lane labeled with M stands for BLUEstain 2 Protein ladder, 5-245 kDa (Goldbio, P008-500). The protein names used for lane labels represent the protein structure from N-terminal to C-terminal order. His, His-Tag; GFP, enhanced green fluorescent protein; 2GS, two GS-linker sequence (GGGGS) repeats; T7 RNAP, T7 RNA polymerase. The bottom image is the original data.

Figure S19
Figure S19 The result of caffeic acid conversion assay 1 NPGA, Hisps, H3H, and Luz were individually expressed in TXTL and mixed with cofactors and substrates (caffeic acid or hispidin). The luminescence was measured at 25˚C for 1 hour, every 5 minutes. While the reaction with hispidin generated light, the reaction with caffeic acid did not. This indicates that the caffeic acid was not converted into hispidin. Control contains TXTLs without enzyme expression.

Figure S20
Figure S20 The result of caffeic acid conversion assay 2. NPGA, Hisps, H3H, and Luz were individually expressed in TXTL. NPGA and Hisps TXTLs were first mixed with cofactors and substrates (caffeic acid or hispidin). The reaction was incubated at 37˚C for 30 minutes to facilitate phosphopanthetheinylation. The aliquot of the reaction was taken for HPLC analysis after this incubation. Then, H3H and Luz TXTLs were added to the reaction and measured the luminescence at 25˚C for 1 hour, every 5 minutes. The reaction with hispidin slightly generated light. The reaction with caffeic acid was as same as Control reaction. This indicates that the caffeic acid was not converted into hispidin. Control stands for a reaction contains TXTLs without enzyme expression. Cloning methods Primer sequences are listed in Table S1. Gene sequences are listed in Table S2. Plasmid names constructed in this paper are summarized in Table S3. Plasmids named with pTXTL contain a carbenicillin resistance gene and plasmids named with pLumi contain an ampicillin resistance gene for antibiotic selection. All the cloned plasmid sequences were verified by sequencing.
In a previous report, in vitro phosphopanthetheinylation assay with NPGA was performed at 37˚C, instead of 30˚C (our TXTL reaction) 33 . Thus, we split the reaction into two steps: (1) Conversion of caffeic acid to hispidin and (2) light generative reaction from hispidin. Briefly, we expressed NPGA, Hisps, H3H, and Luz individually at 30˚C for 8 hours. Then, we mixed NPGA and Hisps TXTLs with 2 mM ATP, 2 mM malonyl CoA, 1 mM CoA, 1 mM NADPH, and 10 mM MgCl 2 . MgCl 2 was added to make the reaction condition similar to the previous in vitro assay 33 . The reaction was incubated at 37˚C for 30 minutes. The aliquot of samples was analyzed by high-performance liquid chromatography (HPLC); however, we did not detect the conversion from caffeic acid to hispidin. We then added H3H and Luz TXTLs to the reaction mixture and measured the luminescence at 25˚C for 1 hour, every 5 minutes. We observed slight luminescence from a reaction containing hispidin as the substrate; however, the reaction containing caffeic acid did not generate any light (Fig. S20).
Lastly, we purified both NPGA and Hisps through His-tagged protein purification and performed an in vitro assay to see the conversion from caffeic acid to hispidin. The 40 µl reaction contained 100 mM Tris-HCl buffer (pH6.8), 1 µM NPGA, 1 µM Hisps, 2 mM ATP, 2 mM malonyl CoA, 500 µM CoA, and 40 mM MgCl 2 . The substrate was either 2 mM caffeic acid or 200 µM hispidin. The reaction was incubated at 37˚C for 30 minutes. After the incubation, we stopped the reaction by adding 200 µl of ethanol, and the liquid was evaporated completely by a speed vacuum concentrator. The pellets were dissolved in methanol to inject HPLC. However, we could not detect the conversion of caffeic acid to hispidin by HPLC.